PROJECT 4 (ROSSO LAB): SUMMARY Post-Traumatic Stress Disorder (PTSD) is a leading cause of global disease burden, and it is twice as common in women as in men. In addition to female sex, genetic factors are established risk factors for PTSD. Furthermore, the diagnosis requires exposure to at least one highly traumatizing stressor, which independently and synergistically with genetic vulnerability leads to long-lasting perturbations in biological arousal systems. A number of moderators of stress responsiveness may contribute to PTSD, including the differential risk seen in women. Altered function of corticotropin releasing factor (CRF) and pituitary adenylate cyclase-activating polypeptide (PACAP) systems represent candidate mechanisms of sex-dependent moderation of PTSD liability. Higher blood PACAP levels and allelic variation in the gene (ADCYAP1R1) encoding the PAC1R receptor predict greater anxious arousal symptoms and total symptoms in women with PTSD, and greater physiological arousal during anxiety-related paradigms. Variation in CRF and its Type-I receptor have also been associated with both PTSD and depression, and sex differences may produce persistent physiological arousal in women exposed to severe stress. Importantly, the neurobiological correlates of CRF/PACAP overlap with arousal-related brain circuitry that is implicated in PTSD, including extended amygdala (extAMG) and medial prefrontal cortex (mPFC). This project is designed to identify CRF-, PACAP-, CRF+PACAP-predominant intermediate phenotypes in women and men with PTSD. Two subregions of the extAMG, the amygdala (AMG) and bed nucleus of the stria terminalis (BNST), will be independently examined, due to evidence of their partially separable roles in fear versus anxiety, and their potentially differential contributions to PTSD. The mPFC will be a focus as a region that modulates arousal responses to threat via reciprocal connections with the AMG and BNST. Using blood samples from adults with DSM-5 PTSD, we will examine CRF and PACAP variation (genetic, epigenetic, mRNA) in relation to markers of hyperarousal across levels of analysis: (1) blood levels of PACAP and hypothalamic pituitary adrenal axis hormones (ACTH and cortisol) as indices of CRF function; (2) resting state functional magnetic resonance imaging of extAMG-mPFC; (4) diffusion tensor imaging of extAMG-mPFC; (5) magnetic resonance spectroscopy of mPFC glutamate metabolism and neuronal integrity (N-acetylaspartate); (6) physiological indices of arousal during fear conditioning and dark enhanced startle paradigms; and (7) clinical symptoms of hyperarousal: anxious arousal, dysphoric arousal, and sleep disruption (using self-report and actigraphy). Project 4 will enhance our understanding of biobehavioral mechanisms of CRF/PACAP in humans, with the potential to identify new PTSD biomarkers. Neuroimaging of BNST function and glutamate metabolism is particularly innovative and may facilitate development of improved diagnostics or therapeutics for individuals with PTSD. We focus on the same peptide systems and brain regions as the other SPARED Projects, and we will use new discoveries from across the Center to continuously refine and optimize our objectives.